Dried and irradiated skin equivalents for ready use

ABSTRACT

The present invention relates generally to systems and methods for preparing, storing, shipping and using skin equivalents made by organotypic culture. In particular, the present invention relates to systems and methods for producing, transporting, storing and using skin equivalents produced by organotypic culture at reduced temperatures, preferably from 2-8 degrees Celsius to ambient temperature. The methods include sterile packaging of the grafts so that the sterility and integrity of the package is maintained until the time of use for grafting purposes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/054,445, filed Oct. 15, 2013, now allowed, which is a divisional ofU.S. patent application Ser. No. 12/612,284, now U.S. Pat. No.8,580,314, filed Nov. 4, 2009, which claims the benefit of expired U.S.Provisional Patent Application No. 61/111,153, filed Nov. 4, 2008, thecontents of all of which are incorporated by reference herein in theirentireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under W81XWH-07-C-0063awarded by the U.S. Army Medical Research and Material Command. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to systems and methods forlong-term storage at refrigerated or ambient temperature of skinequivalents made by organotypic culture.

BACKGROUND

The emerging field of tissue engineering (TE) is poised to make enormousprogress in the treatment of organ disease and dysfunction in the comingdecade. In 2001, there were 23 cell-based therapeutics approved formarket in the United States (U.S.) and Europe, of which nine were skinsubstitutes or grafts, and 100 more products were in development. (DeBree, Genomics-based Drug Data Report and Regenerative Therapy (1)2:77-96 (2001)). In 2007, nearly 100 companies were involved indeveloping engineered tissues, cell-based therapeutics, or relatedtechnologies (Applied Data Research, February 2007). Overall theindustry had an annual growth rate of 16% from 1995-2001. The“structural” industry segment (e.g., skin, bone, cartilage) showed 85%growth from 1998-2001. In 2004, the U.S. market for tissue-engineeredskin replacements/substitutes and active wound repair modulators wasvalued at approximately $195 million. Sales are expected to increase ata compound annual rate of 9.5%, reaching approximately $481 million inthe year 2014 (MedTech Insight, Windhover Information, September 2005).The total U.S. market for advanced wound care technologies was worthmore than $2.3 billion in 2005. This has been projected to grow at anaverage annual growth rate of 12.3% over a five year period to reach$4.6 billion in 2011 (BCC Research, PHM011E, January 2007). The globalwound care market is estimated to be worth U.S.$7.2 billion in 2006 andcomprises two sectors, traditional and advanced (Espicom BusinessIntelligence, 2007). Traditional wound care products consist mainly oflow technology gauze-based dressings such as woven and non-wovensponges, conforming bandages and non-adherent bandages. The advancedwound care segment (US$4.1 billion global) is the fastest growing areawith double-digit growth of 10% per year (Espicom Business Intelligence,2007).

Although a multitude of revolutionary and economically importantapplications for engineered tissues and organs exist in the human healtharena, the full economic potential of the industry is far from realized.At present, only one of the publicly-held tissue engineering companiesworldwide has shown a profit despite global investment in thesetechnologies exceeding $3.5 billion. (Lysaght and Reyes, TissueEngineering 7(5):485-93 (2001)).

A major impediment to the acceptance of engineered tissues by medicalpractitioners, healthcare providers, and second party payers is the lackof a means to effectively and efficiently preserve and store engineeredtissues. The nature of living cells and tissue products makes themimpractical for long-term storage. Current engineered tissues must oftenbe stored and shipped under carefully controlled conditions to maintainviability and function. Typically, engineered tissue products take weeksor months to produce but must be used within hours or days aftermanufacture. As a result, TE companies must continually operate withtheir production facilities at top capacity and absorb the costs ofunsold product which must be discarded. These inventory losses, on topof already costly manufacturing process, have forced prices toimpractical levels. As one specific example, APLIGRAF requires aboutfour weeks to manufacture, is usable for only ten days and must bemaintained between 20 and 23° C. until used. As another example, EPICELis transported by a nurse from Genzyme Biosurgery's production facilityin Cambridge, Mass. to the point of use in a portable incubator and isused immediately upon arrival. Such constraints represent significantchallenges to developing convenient and cost-effective products.

Cryopreservation has been explored as a solution to the storage problem,but it is known to induce tissue damage through ice formation, chillinginjury, and osmotic imbalance. Besides APLIGRAF, the only other approvedliving skin equivalent, ORCEL, is currently in clinical trials as afrozen product but has the drawback that it must be maintained attemperatures below −100° C. prior to use. This requires specializedproduct delivery and storage conditions, including the use of dangerousgoods during transport, and use of liquid nitrogen for storage, which isexpensive, dangerous, and not readily available in rural clinics andfield hospitals. Moreover, delivering a frozen product requires specialtraining on the part of the end user to successfully thaw the tissueprior to use.

Accordingly, what is needed in the art are improved methods of preparingengineered tissues and cells for storage under conditions that areroutinely available at the point of use. As all clinical facilities haverefrigerated storage, development of a skin equivalent that can bestored for prolonged periods in a standard refrigerator would greatlyimprove the availability and clinical utility of these products.Development of a skin equivalent that can be stored for prolongedperiods at ambient temperatures would further increase the availabilityof such products for immediate use on the battlefield or in a variety offirst response situations.

SUMMARY OF THE INVENTION

The present invention relates generally to systems and methods forlong-term storage at refrigerated or ambient temperature of skinequivalents made by organotypic culture. In some embodiments, thepresent invention provides methods of preserving an organotypicallycultured skin equivalent for use as a wound dressing comprising:providing said organotypically cultured skin equivalent and a package;treating said skin equivalent to render cells in the skin equivalentnon-viable; and packaging said skin equivalent to provide a packagedskin equivalent. The present invention is not limited to any particularmethod of treating the skin equivalent to render the cells making up theskin equivalent non-viable. In some embodiments, the treating stepcomprises irradiating said packaged skin equivalent so that said skinequivalent is rendered sterile and non-viable. In some embodiments, theirradiating is performed with gamma irradiation. In some embodiments,the treating step comprises drying said skin equivalent under conditionssuch that cells in said skin equivalent are rendered non-viable. Thepresent invention is not limited to any particular method of drying. Insome embodiments, the drying is performed by a method selected from thegroup consisting of vacuum drying and freeze drying. The presentinvention is not limited to any particular order of steps, unlessotherwise indicated. In some embodiments, the treating occurs beforepackaging. In some embodiments, the treating occurs after packaging. Insome embodiments, the treating comprises drying said skin equivalentunder conditions such that cells making up said skin equivalent arerendered non-viable and irradiating said skin equivalent underconditions such said skin equivalent is rendered sterile. In someembodiments, the drying step occurs before said packaging and saidirradiation step occurs after said packaging step.

The present invention is not limited to the use of any particular skinequivalent. In some embodiments, the organotypically cultured skinequivalent comprises NIKS cells. In some embodiments, the NIKS cellscomprise an exogenous nucleic acid sequence encoding an exogenouspolypeptide. In some embodiments, more than one exogenous polypeptide isexpressed by the cells making up this skin equivalent. The presentinvention is not limited to the use of any particular exogenouspolypeptide. In some embodiments, the exogenous polypeptide is anantimicrobial polypeptide. In some embodiments, the antimicrobialpolypeptide is selected from the group consisting of human beta-defensin1, human beta-defensin 2, human beta-defensin 3, and cathelicidin. Insome embodiments, the antimicrobial polypeptide is provided by the skinequivalent in a quantity of from 1 to 1000 ng of antimicrobialpolypeptide per milliliter of a surface extraction solution, in somepreferred embodiments, the antimicrobial polypeptide is provided by theskin equivalent in a quantity of from 1 to 1000 ng of antimicrobialpolypeptide per milliliter of a surface extraction solution. In someembodiments, the skin equivalent is dried to a final mass of less than75%, 50%, 25% or preferably 15% of that of a wet or non-dried skinequivalent. In some embodiments, the skin equivalent, after rehydration,has an initial DPM value of from about 20 DPM to about 300 DPM,preferably from about 70 to about 140 DPM, and a DPM change value offrom about 5 DPM to about 400 DPM, preferably from about 10 DPM to about220 DPM. In some embodiments, the skin equivalent, after rehydration,has a tensile strength of from about 0.1 to about 5.0 MPa, preferablyfrom about 0.4 to about 1.8 MPa. In some embodiments, the package isheat sealable.

In some embodiments, the present invention provides a packaged humanskin equivalent produced by the foregoing methods. In some embodiments,the present invention provides a packaged, sterile human skin equivalentproduced by the foregoing methods.

In some embodiments, the present invention provides compositionscomprising an isolated, non-viable, in vitro human skin equivalent. Insome embodiments, the skin equivalent is packaged. In some embodiments,the skin equivalent is sterile. In some embodiments, the sterile skinequivalent is irradiated. In some embodiments, the skin equivalent isdried. In some embodiments, the skin equivalent has a mass of less than50% of the mass of a wet skin equivalent. In some embodiments, the skinequivalent comprises NIKS cells. In some embodiments, the NIKS cellscomprise an exogenous nucleic acid sequence encoding an exogenouspolypeptide. In some embodiments, more than one exogenous polypeptide isexpressed by the cells making up the skin equivalent. The presentinvention is not limited to the use of any particular exogenouspolypeptide. In some embodiments, the exogenous polypeptide is anantimicrobial polypeptide. In some embodiments, the antimicrobialpolypeptide is selected from the group consisting of human beta-defensin1, human beta-defensin 2, human beta-defensin 3, and cathelicidin. Insome embodiments, the antimicrobial polypeptide is provided by the skinequivalent in a quantity of from 1 to 1000 ng of antimicrobialpolypeptide per milliliter of a surface extraction solution, in somepreferred embodiments, the antimicrobial polypeptide is provided by theskin equivalent in a quantity of from 1 to 1000 ng of antimicrobialpolypeptide per milliliter of a surface extraction solution. In someembodiments, the skin equivalent is dried to a final mass of less than75%, 50%, 25% or preferably 15% of that of a wet or non-dried skinequivalent. In some embodiments, the skin equivalent, after rehydration,has an initial DPM value of from about 20 DPM to about 300 DPM,preferably from about 70 to about 140 DPM, and a DPM change value offrom about 5 DPM to about 400 DPM, preferably from about 10 DPM to about220 DPM. In some embodiments, the skin equivalent, after rehydration,has a tensile strength of from about 0.1 to about 5.0 MPa, preferablyfrom about 0.4 to about 1.8 MPa.

In some embodiments, the present invention provides methods for treatinga subject comprising providing a skin equivalent composition asdescribed above and applying said skin equivalent to a wound underconditions such that said skin equivalent contacts said wound. In someembodiments, the skin equivalent is applied to said wound temporarily.

In some embodiments, the present invention provides kits comprising apackage containing the skin equivalent composition described above. Insome embodiments, the skin equivalent has a shelf life of from about onemonth to about six months.

In some embodiments, the present invention provides compositionscomprising a nonviable, isolated, in vitro organotypically cultured skinequivalent having a mass of less than 50% of the mass of a wet skinequivalent. In some embodiments, the compositions comprise at least oneexogenous antimicrobial polypeptide expressed by cells integral to saidskin equivalent.

In some embodiments, the present invention provides for the use of theforegoing compositions to treat a subject. In some embodiments, thepresent invention provides for the use of the foregoing compositions totreat a wound on a subject.

DESCRIPTION OF FIGURES

FIG. 1. Viability of tissues post-irradiation. 9F1 and 2D2 tissues wereirradiated, and punch biopsies were harvested at 3, 7, or 14 dayspost-irradiation and analyzed for viability using MTT assay. Datarepresent means+/−the standard deviation measured from at least threeindependent biopsy samples in each treatment group.

FIG. 2. Keratinocyte viability/migration assay (A). Explants whichexhibited no keratinocyte outgrowth were scored negative for viablekeratinocytes. (B). Samples in which keratinocytes had migrated aroundthe edge of the dermis were scored positive for viable keratinocytes.

FIG. 3. Fibroblast outgrowth assay. Biopsies from control and irradiated9F1 tissues were treated with collagenase, and isolated cells werecultured for six days prior to staining with 1% methylene blue tovisualize colonies of cells.

FIG. 4. Protein released from irradiated 2D2 and 9F1 tissues. Human skinsubstitute tissues were irradiated at a dose of 0, 1, or 5 kGy. Proteinswere extracted in water from punch biopsies harvested at 3 dayspost-irradiation and quantified by BCA assay. Data represent meanvalues+/−the standard deviation from four measurements.

FIG. 5. Antimicrobial activity of irradiated 2D2 tissues. Punch biopsiesobtained from non-irradiated and irradiated 2D2 tissues were incubatedfor the indicated times in serum-free culture media. Antimicrobialactivity of material extracted from these tissues was determined by CFUcounting and normalized against control bacterial cultures. Each datapoint represents average values obtained from two independent samples.

FIG. 6. Antimicrobial activity of irradiated 9F1 tissues. Punch biopsiesobtained from non-irradiated and irradiated 9F1 tissues were incubatedfor 4 hr in serum-free culture media. Antimicrobial activity from thesetissues was determined by CFU counts and normalized against controlbacterial cultures, whose value was set to 1. Each data point representsaverage values+/−the standard deviation from four biopsy samples fromtissues in the indicated treatment group.

FIG. 7. Antimicrobial activity of irradiated 2D2 tissues. Punch biopsiesobtained from non-irradiated and irradiated 2D2 tissues were incubatedfor 4 hr in serum-free culture media. Antimicrobial activity from thesetissues was determined by CFU counts and normalized against controlbacterial cultures, whose value was set to 1. Each data point representsaverage values+/−the standard deviation from four biopsy samples fromtissues in the indicated treatment group.

FIG. 8. Total released protein from skin equivalent tissues stored onnutrient gels or nonadherent gauze. Human skin equivalent tissues wereirradiated and punch biopsies were harvested after 14 days and incubatedin 0.2 ml of sterile water at 37° C. for 24 hr. Extracted protein wasquantified by BCA assay. Data represent mean values+/−the standarddeviation from four measurements.

FIG. 9. Histological analysis of freeze-dried irradiated engineered skinequivalents. Fresh skin equivalents were compared to skin equivalentsthat were freeze-dried, or freeze-dried and irradiated at 1 kGy, 5 kGy,or 25 kGy dose level. Tissue sections were stained withhematoxylin/eosin and photographed at 400× magnification. Scale bar=200μm

FIG. 10. Histological analysis of vacuum-dried irradiated engineeredskin equivalents. Fresh skin equivalents were compared to skinequivalents that were vacuum-dried, or vacuum-dried and irradiated at 1kGy, 5 kGy, or 25 kGy dose level. Tissue sections were stained withhematoxylin/eosin and photographed at 400× magnification. Scale bar=200μm

FIG. 11. Viability of irradiated vacuum-dried and freeze-dried skinequivalents. Bar colors represent (from left to right): black=0 kGy(nonirradiated); dark gray=1 kGy; light gray=5 kGy; white=25 kGy. Datapoints represent the average+/−standard deviation (n=4-8), normalized tofreshly prepared, nonirradiated skin equivalent tissue.

FIG. 12. Epidermal barrier function of dried irradiated skinequivalents. Left panel. The change in tissue surface electricalcapacitance was measured over a 10 second interval for freeze-dried orvacuum-dried engineered skin tissues irradiated at 1 kGy, 5 kGy or 25kGy. Values represent mean+/−standard deviation from two measurementsfrom each of two independent tissues. Right panel. Initial DPM valuesare reported for freeze-dried or vacuum-dried tissues irradiated at 1kGy, 5 kGy or 25 kGy. Bar colors represent (from left to right): black=0kGy (nonirradiated); dark gray=1 kGy; light gray=5 kGy; white=25 kGy.Values represent mean+/−standard deviation from two measurements fromeach of two independent tissues.

FIG. 13. Mechanical properties of dried and irradiated engineered skintissues. Bars colors represent (from left to right): black=0 kGy(nonirradiated); dark gray=1 kGy; light gray=5 kGy; white=25 kGy. Dataare mean±std. n=2-4

DEFINITIONS

As used herein, the terms “skin equivalent”, “human skin equivalent”,“human skin substitute”, and “organotypic cultures” are usedinterchangeably to refer to an in vitro derived culture of keratinocytesthat has stratified into squamous epithelia. Typically, the skinequivalents are produced by organotypic culture and include a dermallayer in addition to a keratinocyte layer.

As used herein, the term “wet skin equivalent” refers to a skinequivalent in organotypic culture or immediately removed fromorganotypic culture.

As used herein, the term “non-viable” refers to cells that are notliving as determined by an assay such as an MTT assay.

As used herein, the term “sterile” refers to a skin equivalent that isessentially or completely free of microbial or fungal contamination.

As used herein, the term “dried” refers to a composition from whichmoisture has been removed. A “dried skin equivalent” is a skinequivalent from which moisture has been removed so that the dried skinequivalent has a lower moisture content that a skin equivalent that iswet, or immediately removed from organotypic culture. Comparison of themass of the dried skin equivalent to a wet skin equivalent is used as ameasure of the extent of drying and reflects the amount of moistureremoved from the skin equivalent during the drying process.

As used herein, the term “NIKS cells” refers to cells having thecharacteristics of the cells deposited as cell line ATCC CRL-1219.

The term “homology” refers to a degree of complementarity. There may bepartial homology or complete homology (i.e., identity). A partiallycomplementary sequence is one that at least partially inhibits acompletely complementary sequence from hybridizing to a target nucleicacid and is referred to using the functional term “substantiallyhomologous.” The term “inhibition of binding,” when used in reference tonucleic acid binding, refers to inhibition of binding caused bycompetition of homologous sequences for binding to a target sequence.The inhibition of hybridization of the completely complementary sequenceto the target sequence may be examined using a hybridization assay(Southern or Northern blot, solution hybridization and the like) underconditions of low stringency. A substantially homologous sequence orprobe will compete for and inhibit the binding (i.e., the hybridization)of a completely homologous to a target under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target that lacks even a partial degreeof complementarity (e.g., less than about 30% identity); in the absenceof non-specific binding the probe will not hybridize to the secondnon-complementary target.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of a polypeptideor precursor (e.g., KGF-2). The polypeptide can be encoded by a fulllength coding sequence or by any portion of the coding sequence so longas the desired activity or functional properties (e.g., enzymaticactivity, ligand binding, signal transduction, etc.) of the full-lengthor fragment are retained. The term also encompasses the coding region ofa structural gene and the including sequences located adjacent to thecoding region on both the 5′ and 3′ ends for a distance of about 1 kb oneither end such that the gene corresponds to the length of thefull-length mRNA. The sequences that are located 5′ of the coding regionand which are present on the mRNA are referred to as 5′ untranslatedsequences. The sequences that are located 3′ or downstream of the codingregion and that are present on the mRNA are referred to as 3′untranslated sequences. The term “gene” encompasses both cDNA andgenomic forms of a gene. A genomic form or clone of a gene contains thecoding region interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns maycontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

As used herein, the term “recombinant DNA molecule” refers to a DNAmolecule that is comprised of segments of DNA joined together by meansof molecular biological techniques.

As used herein, the term “purified” or “to purify” refers to the removalof contaminants from a sample.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vehicle” is sometimes used interchangeably with “vector.”

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in prokaryotes usually include a promoter, anoperator (optional), and a ribosome binding site, often along with othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

“Operably linked” refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. A regulatory sequence is “operably linked” to a codingsequence when it is joined in such a way that expression of the codingsequence is achieved under conditions compatible with the regulatorysequence.

The term “transfection” as used herein refers to the introduction offoreign DNA into eukaryotic cells. Transfection may be accomplished by avariety of means known to the art including calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, retroviral infection, and biolistics.

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell thathas stably integrated foreign DNA into the genomic DNA.

The term “transient transfection” or “transiently transfected” refers tothe introduction of foreign DNA into a cell where the foreign DNA failsto integrate into the genome of the transfected cell. The foreign DNApersists in the nucleus of the transfected cell for several days. Duringthis time the foreign DNA is subject to the regulatory controls thatgovern the expression of endogenous genes in the chromosomes. The term“transient transfectant” refers to cells that have taken up foreign DNAbut have failed to integrate this DNA.

As used herein, the term “antimicrobial polypeptide” refers to generallyshort polypeptides, from 5 to 100 amino acids in length, the exhibitantimicrobial activity. Examples of antimicrobial polypeptides include,but are not limited to, human beta-defensins 1, 2, and 3 andcathelicidin. The sequences of a wide variety of antimicrobialpolypeptides within the scope of the invention are known and available,including those identified in WO 05/012,492, incorporated by referenceherein in its entirety.

DETAILED DESCRIPTION

The present invention relates generally to systems and methods forpreparing, shipping and storing skin equivalents made by organotypicculture. In particular, the present invention relates to methods fordrying or irradiating human skin equivalents to eliminate the viabilityof the skin equivalent so that it can be stored for prolonged periodsand transported under standard conditions for use in the field, oron-site use, as opposed to use in a hospital.

Medical planning was a critical part of Operation Iraqi Freedom andincluded predictive models of the expected number of burn casualties(Barillo, D. J., et al., Tracking the daily availability of burn bedsfor national emergencies. J Burn Care Rehabil, 2005. 26(2): p. 174-82).In these models the casualty estimates exceeded the capacity of the onlyDepartment of Defense burn center. The Department of Defense inconjunction with the American Burn Association developed a mass casualtyplan based on the current practices and technology available for burncare. In the first Gulf War the opposing force was known to have usedchemical weapons including sulfur mustard. In the current Iraqi andAfghanistan conflicts, the number of field burns has reached new levels.Cutaneous thermal and chemical vesicant (blistering) burns, as well asthe procedures of deroofing and debridement commonly used to treat theseinjuries, lead to open wounds susceptible to infection by bacterialpathogens.

Unfortunately, in the last 25 years there has been a significant lack ofinnovative, life saving technologies developed for the treatment ofcutaneous burn or vesicant wounds. The need for innovations in this areawas emphasized by the Oct. 25-28, 2006 conference entitled “State of theScience of Burn Research” sponsored by the National Institute of GeneralMedical Sciences. Gamma-irradiated human cadaver skin is stable atambient temperature and has been successfully used in the treatment ofskin defects (Rosales, M. A., M. Bruntz, and D. G. Armstrong,Gamma-irradiated human skin allograft: a potential treatment modalityfor lower extremity ulcers. Int Wound J, 2004. 1(3): p. 201-6; Cancio,L. C., et al., Burn support for Operation Iraqi Freedom and relatedoperations, 2003 to 2004. J Burn Care Rehabil, 2005. 26(2): p. 151-61).However, such products are not indicated for use in wounds that showevidence of infection. Cutaneous wounds, such as those resulting fromvesicant exposure and thermal injuries, provide an ideal environment forbacterial growth and the complications stemming from wound sepsis.Moreover, the increasing frequency of multi-drug resistant clinicalisolates of organisms such as Acinetobacter baumannii, Pseudomonasaeruginosa, and methicillin-resistant Staphylococcus aureus (MRSA)underscores the need for novel approaches to supplement the currentantimicrobial treatment regimes used in cutaneous wound therapy (Milner,S. M. and M. R. Ortega, Reduced antimicrobial peptide expression inhuman burn wounds. Burns, 1999. 25(5): p. 411-3).

In some embodiments, the present invention provides a field-ready,tissue-engineered dried or irradiated antimicrobial skin equivalent fortreatment of vesicant, thermal, and traumatic cutaneous injuries. Thedried or irradiated skin equivalent is designed for long term storage atambient temperatures and maximal versatility and safety to patients withvesicant, thermal, or traumatic injury to external epithelia. Inpreferred embodiments, the dried or irradiated skin equivalents areengineered to deliver the broad spectrum human host defense peptidesβ-defensin-3 (hBD-3) or cathelicidin (hCAP18/LL-37) to the wound bed.

Accordingly, in some embodiments, the present invention provides a driedor irradiated human skin equivalent comprising non-viable cells. In someembodiments, the skin equivalent has been engineered to express andprovide exogenous antimicrobial polypeptides, preferably humanβ-defensin-1, 2 or 3 or cathelicidin (hCAP18/LL37). In some embodiments,the non-viable skin equivalents are applied to wounds. In someembodiments, the non-viable human skin equivalents are appliedtemporarily to wounds. In some embodiments, the non-viable human skinequivalents are removed and replaced with additional non-viable humanskin equivalents providing the same antimicrobial polypeptide. In otherembodiments, the non-viable skin equivalents are removed and replacedwith additional non-viable skin equivalents providing a differentantimicrobial polypeptide. In other embodiments non-viable human skinequivalents are removed prior to application of a viable skin equivalentor a permanent skin graft on the wound (e.g., burn wound).

In preferred embodiments, the skin equivalents of the present inventionare engineered to express an exogenous antimicrobial polypeptide. Thepresent invention is not limited to the use of any particularantimicrobial polypeptide. In preferred embodiments, the antimicrobialpolypeptide is human β-defensin-1, human β-defensin-2, humanβ-defensin-3, or cathelicidin (hCAP-18/LL37) or variant. In somepreferred embodiments, nucleic acid constructs or vectors encoding theantimicrobial polypeptide are introduced into the keratinocytes (e.g.,NIKS cells) and the transfected keratinocytes are used to make the skinequivalent by organotypic culture techniques. Preferred embodiments forthe production of skin equivalents expressing exogenous polypeptides, aswell as additional wild-type and variant antimicrobial polypeptides, areprovided in co-pending application Ser. No. 10/909,119, the entirecontents of which are incorporated herein by reference.

A) Skin Equivalents Produced by Organotypic Culture

The present invention is not limited to the use of any particular sourceof cells that are capable of differentiating into squamous epithelia.Indeed, the present invention contemplates the use of a variety of celllines and sources that can differentiate into squamous epithelia,including both primary and immortalized keratinocytes. Sources of cellsinclude keratinocytes and dermal fibroblasts biopsied from humans andcavaderic donors (Auger et al., In Vitro Cell. Dev. Biol.—Animal36:96-103; U.S. Pat. Nos. 5,968,546 and 5,693,332, each of which isincorporated herein by reference), neonatal foreskins (Asbill et al.,Pharm. Research 17(9): 1092-97 (2000); Meana et al., Burns 24:621-30(1998); U.S. Pat. Nos. 4,485,096; 6,039,760; and 5,536,656, each ofwhich is incorporated herein by reference), and immortalizedkeratinocytes cell lines such as NM1 cells (Baden, In Vitro Cell. Dev.Biol. 23(3):205-213 (1987)), HaCaT cells (Boucamp et al., J. cell. Boil.106:761-771 (1988)); and NIKS cells (Cell line BC-1-Ep/SL; U.S. Pat. No.5,989,837, incorporated herein by reference; ATCC CRL-12191). Each ofthese cell lines can be cultured or genetically modified in order toproduce a cell line capable of expressing or co-expressing the desiredprotein(s). In particularly preferred embodiments, NIKS cells areutilized. The discovery of a novel human keratinocyte cell line(near-diploid immortalized keratinocytes or NIKS) provides anopportunity to genetically engineer human keratinocytes with non-viralvectors. A unique advantage of the NIKS cells is that they are aconsistent source of genetically-uniform, pathogen-free humankeratinocytes. For this reason, they are useful for the application ofgenetic engineering and genomic gene expression approaches to provideskin equivalent cultures with enhanced properties over currentlyavailable technologies and skin tissue products. The NIKS keratinocytecell line, identified and characterized at the University of Wisconsin,is nontumorigenic, exhibits a stable karyotype, and exhibits normalgrowth and differentiation both in monolayer and organotypic culture.NIKS cells form fully stratified skin equivalents in culture. Thesecultures are indistinguishable by all criteria tested thus far fromorganotypic cultures formed from primary human keratinocytes. Unlikeprimary cells however, the immortalized NIKS cells will continue toproliferate in monolayer culture indefinitely. This provides anopportunity to genetically manipulate the cells and isolate new clonesof cells with new useful properties (Allen-Hoffmann et al., J. Invest.Dermatol., 114(3): 444-455 (2000)).

The NIKS cells arose from the BC-1-Ep strain of human neonatal foreskinkeratinocytes isolated from an apparently normal male infant. In earlypassages, the BC-1-Ep cells exhibited no morphological or growthcharacteristics that were atypical for cultured normal humankeratinocytes. Cultivated BC-1-Ep cells exhibited stratification as wellas features of programmed cell death. To determine replicative lifespan,the BC-1-Ep cells were serially cultivated to senescence in standardkeratinocyte growth medium at a density of 3×10⁵ cells per 100-mm dishand passaged at weekly intervals (approximately a 1:25 split). Bypassage 15, most keratinocytes in the population appeared senescent asjudged by the presence of numerous abortive colonies which exhibitedlarge, flat cells. However, at passage 16, keratinocytes exhibiting asmall cell size were evident. By passage 17, only the small-sizedkeratinocytes were present in the culture and no large, senescentkeratinocytes were evident. The resulting population of smallkeratinocytes that survived this putative crisis period appearedmorphologically uniform and produced colonies of keratinocytesexhibiting typical keratinocyte characteristics including cell-celladhesion and apparent squame production. The keratinocytes that survivedsenescence were serially cultivated at a density of 3×10⁵ cells per100-mm dish. Typically the cultures reached a cell density ofapproximately 8×10⁶ cells within 7 days. This stable rate of cell growthwas maintained through at least 59 passages, demonstrating that thecells had achieved immortality. The keratinocytes that emerged from theoriginal senescencing population were originally designatedBC-1-Ep/Spontaneous Line and are now termed NIKS. The NIKS cell line hasbeen screened for the presence of proviral DNA sequences for HIV-1,HIV-2, EBV, CMV, HTLV-1, HTLV-2, HBV, HCV, B-19 parvovirus, HPV-16,SV40, HHV-6, HHV-7, HPV-18 and HPV-31 using either PCR or Southernanalysis. None of these viruses were detected.

Chromosomal analysis was performed on the parental BC-1-Ep cells atpassage 3 and NIKS cells at passages 31 and 54. The parental BC-1-Epcells have a normal chromosomal complement of 46, XY. At passage 31, allNIKS cells contained 47 chromosomes with an extra isochromosome of thelong arm of chromosome 8. No other gross chromosomal abnormalities ormarker chromosomes were detected. The karyotype of the NIKS cells hasbeen shown to be stable to at least passage 54.

The DNA fingerprints for the NIKS cell line and the BC-1-Epkeratinocytes are identical at all twelve loci analyzed demonstratingthat the NIKS cells arose from the parental BC-1-Ep population. The oddsof the NIKS cell line having the parental BC-1-Ep DNA fingerprint byrandom chance is 4×10⁻¹⁶. The DNA fingerprints from three differentsources of human keratinocytes, ED-1-Ep, SCC4 and SCC13y are differentfrom the BC-1-Ep pattern. This data also shows that keratinocytesisolated from other humans, ED-1-Ep, SCC4, and SCC13y, are unrelated tothe BC-1-Ep cells or each other. The NIKS DNA fingerprint data providesan unequivocal way to identify the NIKS cell line.

Loss of p53 function is associated with an enhanced proliferativepotential and increased frequency of immortality in cultured cells. Thesequence of p53 in the NIKS cells is identical to published p53sequences (GenBank accession number: M14695). In humans, p53 exists intwo predominant polymorphic forms distinguished by the amino acid atcodon 72. Both alleles of p53 in the NIKS cells are wild-type and havethe sequence CGC at codon 72, which codes for an arginine. The othercommon form of p53 has a proline at this position. The entire sequenceof p53 in the NIKS cells is identical to the BC-1-Ep progenitor cells.Rb was also found to be wild-type in NIKS cells.

Anchorage-independent growth is highly correlated to tumorigenicity invivo. For this reason, the anchorage-independent growth characteristicsof NIKS cells in agar or methylcellulose-containing medium wereinvestigated. NIKS cells remained as single cells after 4 weeks ineither agar- or methylcellulose-containing medium. The assays werecontinued for a total of 8 weeks to detect slow growing variants of theNIKS cells. None were observed.

To determine the tumorigenicity of the parental BC-1-Ep keratinocytesand the immortal NIKS keratinocyte cell line, cells were injected intothe flanks of athymic nude mice. The human squamous cell carcinoma cellline, SCC4, was used as a positive control for tumor production in theseanimals. The injection of samples was designed such that animalsreceived SCC4 cells in one flank and either the parental BC-1-Epkeratinocytes or the NIKS cells in the opposite flank. This injectionstrategy eliminated animal to animal variation in tumor production andconfirmed that the mice would support vigorous growth of tumorigeniccells. Neither the parental BC-1-Ep keratinocytes (passage 6) nor theNIKS keratinocytes (passage 35) produced tumors in athymic nude mice.

NIKS cells were analyzed for the ability to undergo differentiation inboth submerged culture and organotypic culture. Techniques fororganotypic culture are described in detail in the examples. Inparticularly preferred embodiments, the organotypically cultured skinequivalents of the present invention comprise a dermal equivalent formedfrom collagen or a similar material and fibroblasts. The keratinocytes,for example NIKS cells or a combination of NIKS cells and cells from apatient are seeded onto the dermal equivalent and form an epidermallayer characterized by squamous differentiation following theorganotypic culture process.

For cells in submerged culture, the formation cornified envelopes wasmonitored as a marker of squamous differentiation. In cultured humankeratinocytes, early stages of cornified envelope assembly result in theformation of an immature structure composed of involucrin, cystatin-αand other proteins, which represent the innermost third of the maturecornified envelope. Less than 2% of the keratinocytes from the adherentBC-1-Ep cells or the NIKS cell line produce cornified envelopes. Thisfinding is consistent with previous studies demonstrating that activelygrowing, subconfluent keratinocytes produce less than 5% cornifiedenvelopes. To determine whether the NIKS cell line is capable ofproducing cornified envelopes when induced to differentiate, the cellswere removed from adherent culture and suspended for 24 hours in mediummade semi-solid with methylcellulose. Many aspects of terminaldifferentiation, including differential expression of keratins andcornified envelope formation can be triggered in vitro by loss ofkeratinocyte cell-cell and cell-substratum adhesion. The NIKSkeratinocytes produced as many as and usually more cornified envelopesthan the parental keratinocytes. These findings demonstrate that theNIKS keratinocytes are not defective in their ability to initiate theformation of this cell type-specific differentiation structure.

To confirm that the NIKS keratinocytes can undergo squamousdifferentiation, the cells were cultivated in organotypic culture.Keratinocyte cultures grown on plastic substrata and submerged in mediumreplicate but exhibit limited differentiation. Specifically, humankeratinocytes become confluent and undergo limited stratificationproducing a sheet consisting of 3 or more layers of keratinocytes. Bylight and electron microscopy there are striking differences between thearchitecture of the multilayered sheets formed in submerged culture andintact human skin. In contrast, organotypic culturing techniques allowfor keratinocyte growth and differentiation under in vivo-likeconditions. Specifically, the cells adhere to a physiological substratumconsisting of dermal fibroblasts embedded within a fibrillar collagenbase. The organotypic culture is maintained at the air-medium interface.In this way, cells in the upper sheets are air-exposed while theproliferating basal cells remain closest to the gradient of nutrientsprovided by diffusion through the collagen gel. Under these conditions,correct tissue architecture is formed. Several characteristics of anormal differentiating epidermis are evident. In both the parental cellsand the NIKS cell line a single layer of cuboidal basal cells rests atthe junction of the epidermis and the dermal equivalent. The roundedmorphology and high nuclear to cytoplasmic ratio is indicative of anactively dividing population of keratinocytes. In normal humanepidermis, as the basal cells divide they give rise to daughter cellsthat migrate upwards into the differentiating layers of the tissue. Thedaughter cells increase in size and become flattened and squamous.Eventually these cells enucleate and form cornified, keratinizedstructures. This normal differentiation process is evident in the upperlayers of both the parental cells and the NIKS cells. The appearance offlattened squamous cells is evident in the upper epidermal layers anddemonstrates that stratification has occurred in the organotypiccultures. In the uppermost part of the organotypic cultures theenucleated squames peel off the top of the culture. To date, nohistological differences in differentiation at the light microscopelevel between the parental keratinocytes and the NIKS keratinocyte cellline grown in organotypic culture have been observed.

To observe more detailed characteristics of the parental (passage 5) andNIKS (passage 38) organotypic cultures and to confirm the histologicalobservations, samples were analyzed using electron microscopy. Parentalcells and the immortalized NIKS human keratinocyte cell line wereharvested after 15 days in organotypic culture and sectionedperpendicular to the basal layer to show the extent of stratification.Both the parental cells and the NIKS cell line undergo extensivestratification in organotypic culture and form structures that arecharacteristic of normal human epidermis. Abundant desmosomes are formedin organotypic cultures of parental cells and the NIKS cell line. Theformation of a basal lamina and associated hemidesmosomes in the basalkeratinocyte layers of both the parental cells and the cell line wasalso noted.

Hemidesmosomes are specialized structures that increase adhesion of thekeratinocytes to the basal lamina and help maintain the integrity andstrength of the tissue. The presence of these structures was especiallyevident in areas where the parental cells or the NIKS cells had attacheddirectly to the porous support. These findings are consistent withearlier ultrastructural findings using human foreskin keratinocytescultured on a fibroblast-containing porous support. Analysis at both thelight and electron microscopic levels demonstrate that the NIKS cellline in organotypic culture can stratify, differentiate, and formstructures such as desmosomes, basal lamina, and hemidesmosomes found innormal human epidermis.

B) Drying or Irradiation of Skin Equivalents

In preferred embodiments, the skin equivalents produced as described inExample 1 are irradiated and/or dried to provide a non-viable skinequivalent. In some embodiments, the skin equivalents are dried toeliminate cell viability. In some embodiments, the skin equivalents areirradiated, for example, gamma irradiated. In some embodiments, the skinequivalents are dosed with from about 0.5 kGy to about 25 kGy gammaradiation. In some embodiments, the skin equivalents are dosed fromabout 0.5 to about 12 kGy of radiation, more preferably from about 1 kGyto about 5 kGy gamma irradiation. In any event, the amount of radiationdelivered to the skin equivalent is preferably enough to cause the cellscontained in the skin equivalent to be non-viable as assayed by an MTTviability assay or other appropriate viability assays. In furtherpreferred embodiments, the skin equivalents are dried under vacuum orfreeze dried. In some embodiments, the skin equivalents are dried beforeirradiation. In some preferred embodiments, the skin equivalents arepackaged in a sterile package prior to drying or irradiation. In furtherpreferred embodiments, the skin equivalents are packaged with a sterilefabric such as gauze to permit storage, transport, and ease of use bythe end user. In other embodiments, the dried or irradiated skinequivalents maintain the ability to release endogenous polypeptides tothe surface of a wound after being contacted with a wound. In furtherembodiments, the dried or irradiated skin equivalents maintain theability to release exogenous polypeptides to the wound after beingcontacted with the wound environment. In further embodiments, the skinequivalents are refrigerated prior to use, while in other embodiments,the skin equivalents are stored at ambient temperatures prior to use.

It will be recognized that the extent to which the skin equivalent hasbeen dried can be determined by comparing the mass of the dried skinequivalent to the mass of a skin equivalent that has not been dried (awet skin equivalent), i.e., a skin equivalent that has just been removedfrom organotypic culture. In some embodiments, the skin equivalent isdried to a final mass of less than 75%, 50%, 25% or preferably 15% ofthat of the wet skin equivalent. In some embodiments, the dried skinequivalents of the present invention have a mass of less than 75%, 50%,25% or preferably 15% of that of a wet or non-dried skin equivalent. Insome embodiments, the dried skin equivalents are rehydrated prior toapplication to a subject. In some embodiments, the rehydrated skinequivalents have a tensile strength of from 0.1 to 5.0 MPa, preferablyfrom about 0.4 to about 1.8 MPa. In some embodiments, the rehydratedskin equivalents have an initial DPM value of from about 20 DPM to about300 DPM, preferably from about 70 to about 140 DPM, and a DPM changevalue of from about 5 DPM to about 400 DPM, preferably from about 10 DPMto about 220 DPM.

In some embodiments, the dried and/or irradiated skin equivalents areutilized for delivery of a peptide or protein of interest to a subject,and in some preferred embodiments to a wound bed on a subject. Skinequivalents that express exogenous peptides and proteins have beenpreviously described by the inventors, see, e.g., WO 05/012492,incorporated herein by reference in its entirety. In some embodiments,the skin equivalents are engineered to express one or antimicrobialpolypeptides. In some embodiments, the antimicrobial polypeptide iscathelicidin, human beta-defensin 1, human beta-defensin 2, or humanbeta-defensin 3, or combinations thereof. In preferred embodiments, thepeptide or polypeptide is exogenous, i.e., encoded and expressed by anexogenous gene construct engineered into the keratinocytes utilized tomake the skin equivalent. The amount of peptide or polypeptide deliveredby the skin equivalent can be determined by applying an aqueous solutionto the skin equivalent and measuring the amount of peptide orpolypeptide that is delivered into the solution. In some embodiments,the polypeptide is provided in a quantity of from 1 to 1000 ng ofantimicrobial polypeptide per milliliter of a extraction solution. Insome embodiments, the polypeptide is provided in a quantity of from 10to 500 ng of antimicrobial polypeptide per milliliter of an extractionsolution.

C) Therapeutic Uses

It is contemplated that the non-viable skin equivalents of the presentinvention may be used therapeutically. In some embodiments, the dried orirradiated skin is used in wound closure and burn treatmentapplications. The use of autografts and allografts for the treatment ofburns and wound closure is described in Myers et al., A. J. Surg.170(1):75-83 (1995) and U.S. Pat. Nos. 5,693,332; 5,658,331; and6,039,760, each of which is incorporated herein by reference. In someembodiments, the skin equivalents may be used in conjunction with dermalreplacements such as DERMAGRAFT or INTEGRA. Accordingly, the presentinvention provides methods for wound closure, including wounds caused byburns, comprising providing a skin equivalent and a patient sufferingfrom a wound and treating the patient with the skin equivalent underconditions such that the wound is closed.

In some embodiments, the skin equivalents are utilized to treat chronicskin wounds. Chronic skin wounds (e.g., venous ulcers, diabetic ulcers,pressure ulcers) are a serious problem. The healing of such a woundoften takes well over a year of treatment. Treatment options currentlyinclude dressings and debridement (use of chemicals or surgery to clearaway necrotic tissue), and/or antibiotics in the case of infection.These treatment options take extended periods of time and high amountsof patient compliance. As such, a therapy that can increase apractitioner's success in healing chronic wounds and accelerate the rateof wound healing would meet an unmet need in the field. Accordingly, thepresent invention contemplates treatment of skin wounds with skinequivalents comprising the cells of the present invention (e.g., NIKScells). In some embodiments, skin equivalents are topically applied towounds. In other embodiments, skin equivalents comprising NIKS cells areused for engraftment on partial thickness wounds. In other embodiments,skin equivalents comprising NIKS cells are used for engraftment on fullthickness wounds. In other embodiments, skin equivalents comprising NIKScells are used to treat numerous types of internal wounds, including,but not limited to, internal wounds of the mucous membranes that linethe gastrointestinal tract, ulcerative colitis, and inflammation ofmucous membranes that may be caused by cancer therapies. In still otherembodiments, skin equivalents comprising NIKS cells expressing hostdefense peptides are used as a temporary or permanent wound dressing.

In still further embodiments, the cells are engineered to provideadditional therapeutic agents to a subject. The present invention is notlimited to the delivery of any particular therapeutic agent. Indeed, itis contemplated that a variety of therapeutic agents may be delivered tothe subject, including, but not limited to, enzymes, peptides, peptidehormones, other proteins, ribosomal RNA, ribozymes, small interferingRNA (siRNA) micro RNA (miRNA), and antisense RNA. In preferredembodiments, the agents are host defense peptides such as humanbeta-defensin 1, 2, or 3 or cathelicidin, see, e.g., U.S. patentapplication Ser. No. 10/909,119, incorporated herein by reference in itsentirety. These therapeutic agents may be delivered for a variety ofpurposes, including but not limited to the purpose of correcting geneticdefects. In some particular preferred embodiments, the therapeutic agentis delivered for the purpose of detoxifying a patient with an inheritedinborn error of metabolism (e.g., aminoacidopathesis) in which the graftserves as wild-type tissue. It is contemplated that delivery of thetherapeutic agent corrects the defect. In some embodiments, the cellsare transfected with a DNA construct encoding a therapeutic agent (e.g.,insulin, clotting factor IX, erythropoietin, etc) and the transfectedcells are administered to the subject. The therapeutic agent is thendelivered to the patient's bloodstream or other tissues from the graft.In preferred embodiments, the nucleic acid encoding the therapeuticagent is operably linked to a suitable promoter. The present inventionis not limited to the use of any particular promoter. Indeed, the use ofa variety of promoters is contemplated, including, but not limited to,inducible, constitutive, tissue-specific, and keratinocyte-specificpromoters. In some embodiments, the nucleic acid encoding thetherapeutic agent is introduced directly into the keratinocytes (i.e.,by electroporation, calcium phosphate co-precipitation, or liposometransfection). In other preferred embodiments, the nucleic acid encodingthe therapeutic agent is provided as a vector and the vector isintroduced into the keratinocytes by methods known in the art. In someembodiments, the vector is an episomal vector such as a replicatingplasmid. In other embodiments, the vector integrates into the genome ofthe keratinocytes. Examples of integrating vectors include, but are notlimited to, retroviral vectors, adeno-associated virus vectors,non-replicating plasmid vectors and transposon vectors.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); mM (millimolar); μM(micromolar); N (Normal); mol (moles); mmol (millimoles); μmol(micromoles); nmol (nanomoles); g (grams); mg (milligrams); μg(micrograms); ng (nanograms); l or L (liters); ml or mL (milliliters);μl or μL (microliters); cm (centimeters); mm (millimeters); μm(micrometers); nm (nanometers); C (degrees Centigrade); U (units), mU(milliunits); min. (minutes); sec. (seconds); % (percent); kb(kilobase); bp (base pair); PCR (polymerase chain reaction); BSA (bovineserum albumin); CFU (colony forming units); kGy (kiloGray); PVDF(polyvinylidine fluoride); BCA (bicinchoninic acid); SDS-PAGE (sodiumdodecyl sulfate polyacrylamide gel electrophoresis).

Example 1

This example describes a method for the production of skin equivalents.

Media. The organotypic culture process uses three different culturemedia, all based on the formulation of SMB medium described in U.S. Pat.No. 7,407,805, with the exception that cholera toxin is omitted from allmedia. FM01 is used to propagate the normal human dermal fibroblasts(NHDFs) for use in skin equivalent dermal equivalent layers. FM01 hasthe same formulation as SMB except that it contains Fetal Clone II serum(2% final) and lacks cholera toxin. KM01 is used to grow NIKSkeratinocytes and has the same composition as SMB except that itcontains 2.5% fetal clone II, and additional epidermal growth factor(EGF) is added to a final concentration of 5 ng/ml. SM01 is used duringthe epidermal stratification phase of skin equivalent production and isidentical to SMB except for the omission of cholera toxin.

Dermal equivalent preparation. On day 0, frozen NHDF cells are thawedand plated. The cells are fed FM01 the next day (day 1) to removeresidual cryoprotectant and again on day 3. On day 4, they are harvestedfor use in the dermal equivalent. To prepare the dermal equivalent, TypeI rat-tail collagen is first diluted to 3 mg/ml in 0.03N acetic acid andchilled on ice. A mixture of concentrated Ham's F12 medium (8.7× normalstrength and buffered with HEPES at pH 7.5) is mixed with fetal cloneII. These two solutions are 11.3 and 9.6% of the final solution volume.1N NaOH is added to the medium mixture (2.4% of final solution). Thediluted collagen is then added (74.7%) to the mixture. A 2% volume ofsuspended fibroblasts (2.78×10⁶/ml) is added to the mixture. 9 ml of thefinal dermal equivalent mixture is poured into each 75 mm TRANSWELLinsert (Corning Costar). After a 50-70 minute gel formation period, theTranswell inserts are transferred to the surface of a stainless steelmesh in a 150 mm culture dish. 80 ml of FM01 is placed in the 150 mmdish outside the TRANSWELL insert and 10 ml is placed on top of thedermal equivalent. The dermal equivalents are placed in 37° C., 5% CO₂,90% relative humidity incubator for 4-5 days prior to use in theorganotypic cultures.

NIKS Growth and Seeding. NIKS cells are thawed and plated at a densityof approximately 5×10⁵ cells per 100 mm dish. NIKS culture can beperformed in the presence or absence of murine feeder cells. On day 1,the NIKS cells are fed fresh KM01 to remove residual cryoprotectant. TheNIKS cells are fed again on day 3. On day 4, the NIKS cells areharvested from the initial p100 cultures and seeded into 225 cm² cultureflasks at a density of 1.2×10⁶ per flask. The NIKS cultures are fedfresh medium on Days 7 and 8. On day 9, the NIKS cells are harvested,counted, and resuspended in SM01. 2.27×10⁴ NIKS cells/cm² are seededonto the surface of the dermal equivalents. The dishes are cultures arefed and lifted to the air-medium interface. Cultures are transferred toa controlled humidity incubator set to 75% where they remain for therest of their growth. Cultures are fed SM01 on days 14, 18, 22, 25, 28,and 30.

Example 2 Determination of the Lethal Dose of Gamma Radiation Requiredto Produce Nonviable, Sterile Skin Equivalent Tissue

The production schedule for development of irradiated skin equivalentswas based on the 28-day production process previously established forengineered human skin tissue products. 9F1 and 2D2 tissues, which hadbeen engineered for enhanced expression of the host defense peptideshBD-3 and hCAP18/LL-37, respectively, were produced using asepticprocedures. Tissues were transferred onto nutrient gel chambers andsealed prior to packaging and transport. Gamma irradiation was performedby Sterigenics, Inc. using the ExCell high precision gamma irradiator,at its Charlotte, N.C. facility. Tissues received one of five radiationdoses: 0 kGy, 1 kGy, 5 kGy, 8 kGy, or 11 kGy. These doses were chosenbased on previous evaluation of similar doses on allograft tissues.After irradiation, tissues were refrigerated prior to analysis at thetimes indicated below.

Upon return of processed samples to Stratatech, it was found that sometissues stored on nutrient gels were detached from their underlyingsupport membranes, resulting in folding and wrinkling of tissues. Thisphenomenon was observed in both irradiated and nonirradiated tissues.Shipping procedures were subsequently developed that solve this problem.See Examples below.

Sterility testing: Punch biopsies were obtained from 9F1 and 2D2 tissuesthat had been subjected to gamma irradiation at doses ranging from 0 to11 kGy. Samples from tissues stored for 3 days post-irradiation wereinoculated into trypticase soy broth or fluid thioglycolate media.Cultures were incubated for 14 days under conditions defined forsterility testing by the US Pharmacopeia. After 14 days, cultures werevisually examined for microbial growth. No microbial growth was observedfor any of the analyzed tissues, demonstrating the tissues remainedsterile throughout handling, irradiation, and transport.

Viability testing by MTT viability assay: Biopsies were obtained fromcontrol or gamma irradiated 9F1 and 2D2 tissues at 3 days, 7 days, or 14days after irradiation treatment and cell viability was measured by MTTviability assay. Briefly, the MTT substrate,3-(4,5-dimethylthiazolyl-2-yl)-2, 5-diphenyl tetrazolium bromide, isconverted to MTT formazan product by cellular dehydrogenases in viablecells. The colored product is then extracted into isopropanol and readat 550 nm. Viability was measured in four independent tissue batches,and representative results are shown in FIG. 1. The viability ofunirradiated tissues did not change significantly during the storageperiod. Tissues treated with 1 kGy radiation had residual enzymaticactivity at 3 days post-irradiation that continued to decrease at 7 and14 days post-irradiation. However, tissues treated with 5, 8, or 11 kGydoses demonstrated minimal residual activity at all timepoints andremained low throughout the course of the 14-day storage period. By 14days after irradiation, the metabolic activity of tissues irradiatedwith 1 kGy was reduced to the same low level seen with higher radiationdoses.

Viability determination by keratinocyte migration assay: Biopsy explantcultures were established from control tissues or irradiated tissues todetermine if keratinocytes were inactivated by radiation treatments.Punch biopsies obtained from tissues at 3 days post irradiation weretransferred to growth media, cultured for 48 hours, and processed forhistological staining using hematoxylin/eosin and digital microscopy.Images were obtained to assess tissue architecture and scored aspositive or negative for keratinocyte migration out from wound edgescreated by biopsy punches according to the schematic in FIG. 2. Cellmigration was evident in nonirradiated 9F1 and 2D2 tissues but wasabsent from all tissues subjected to gamma irradiation.

Fibroblast outgrowth assay: Biopsies were obtained from control andirradiated tissues at 7 days post-irradiation, and were treated withbacterial collagenase to release fibroblasts. Cells liberated from thesetissues were transferred to culture plates, allowed to grow for six daysin culture media, and visualized by methylene blue staining. Fibroblastcell outgrowth was scored as positive or negative based on the presenceor absence of stained blue cells, respectively. Images of fibroblastcultures isolated from non-irradiated or irradiated tissues are shown inFIG. 3. Cell outgrowth was observed from control tissues receiving noradiation, but was absent from preparations derived from irradiatedtissues.

From the above studies, it was determined that product sterility wasmaintained throughout production and processing of tissues. Treatment ofskin equivalent tissues with radiation doses of 1 kGy yielded nonviabletissue using two independent assays of viability. Doses of 5 kGy orhigher resulted in nonviable tissue as assessed by all three methods.

Example 3 Evaluation of the Structural Properties of Gamma-IrradiatedSkin Equivalent Tissue

The structural properties of irradiated tissues after 3 days ofrefrigerated storage were evaluated by histological staining withhematoxylin and eosin and visualized by light microscopy. Histologicalstaining verified that irradiated tissues retain normal tissueorganization and gross morphology. All cellular layers, includingdermis, basal and spinous keratinocyte layers, and stratum corneum, wereidentifiable in nonirradiated control and irradiated tissues, thoughsome tissue damage was apparent at higher radiation doses, visualized asgaps between the dermal and epidermal compartments. As a result, 1 kGyand 5 kGy dose levels were identified as the most promising for use insubsequent studies.

Example 4 Evaluation of the Biochemical Properties, Including the Invitro Antimicrobial Activity, of Gamma-Irradiated Skin Equivalent Tissue

Gamma radiation stimulates dose-dependent damage to proteins bothdirectly and indirectly. Direct damage is initiated through ionization,while indirect damage involves hydrolysis of water molecules andoxidative modification or crosslinking of macromolecules. As a result,tissue protein may exhibit increased degradation and reduced solubilityupon irradiation. Since the product under development is expected tofunction through provision of elevated levels of host defense peptides,it was necessary to determine protein accessibility and biologicalactivity after irradiation. Analysis of irradiated tissues includedsoluble and total protein analysis, immunoblot analysis, andantimicrobial activity assays.

Soluble and total protein analysis: Punch biopsies were obtained fromnon-irradiated and irradiated 2D2 and 9F1 skin tissues and submerged in0.2 mL sterile water. Biopsy samples were incubated at 37° C. for 72 hrto extract protein, supernatants were collected, and total proteinquantified by BCA assay. As shown in FIG. 4, total protein eluted intothe supernatant decreased with increasing radiation dose. These resultsare consistent with widely reported radiation-mediated damage ofproteins by crosslinking, a modification that reduces proteinsolubility. However, in preliminary studies using tissues packaged in aless hydrated environment consisting of nonadherent gauze, thisradiation dose dependent decrease in protein extractability waseliminated (see Example 6).

Peptide analysis: Protein extractability may reflect the generalbiochemical state of processed tissues; more relevant parameterssupporting the development of an antimicrobial wound dressing are theintegrity and solubility of antimicrobial peptides. To evaluate theseparameters, immunoblot analysis was performed on proteins extracted fromirradiated and non-irradiated 2D2 tissues. Punch biopsies were obtainedfrom 2D2 tissues and transferred into serum-free growth medium for anadditional 48 hr. Conditioned media from duplicate tissues werecollected, proteins were resolved by SDS-PAGE, transferred to PVDFmembranes, and processed for immunoblot analysis using standard methods.Antimicrobial peptides were detected using hCAP18-specific antibodies,which detect the intact human antimicrobial protein hCAP18 and itsbioactive proteolytic fragment, LL-37. Both intact hCAP18 and LL-37 werereadily detected in media conditioned by tissues, independent ofradiation dose, and the migration pattern of these proteins on SDS-PAGEwas also unaffected by radiation doses utilized in these studies. Therewere slight increases in peptide released from non-irradiated controltissues. These slight increases may be due to new synthesis andsecretion of peptide from the viable tissue, or as a result of increasedpeptide solubility in these tissues. Despite this observation,cathelicidin antimicrobial peptides remain intact and can be readilyextracted from irradiated tissues.

Antimicrobial activity assay: Having established the integrity ofreleased antimicrobial peptides from non-irradiated and irradiatedtissues, we evaluated control and irradiated tissues for antimicrobialproperties. Briefly, punch biopsies were obtained from irradiated ornon-irradiated tissues, and transferred to serum-free culture medium for2, 4, or 24 hr. to allow for extraction of antimicrobial peptides. Mediawere collected and combined with an inoculum of 1.0×10³ CFU of S.carnosus in bacterial growth media. This mixture was incubated at 37° C.with constant shaking for 60 min. Afterward, samples were plated ontobacteriological plates using a WASP2 Spiral Plater (MicrobiologyInternational, Frederick, Md.) and incubated for 16 hr. at 37° C.Colonies were counted, and viable bacterial density, expressed inCFU/mL, was determined. Values were normalized to the density ofbacterial cultures grown in the presence of serum-free growth mediumwithout tissue extracts. As shown in FIG. 5, samples extracted from 2D2tissues treated with 1 kGy radiation exhibited enhanced antimicrobialactivity as indicated by decrease in bacterial density relative tonon-irradiated tissues at conditioning periods of 2 and 4 hours. Thistransient increase in antimicrobial activity was not observed in tissuestreated with a 5 kGy dose, nor was it evident under longer tissueconditioning times. A similar increase in activity was seen in 9F1tissues that had been irradiated at 1 kGy (data not shown). Thisimproved antimicrobial activity is surprising given that irradiatedtissues may release lower amounts of total protein and cathelicidinantimicrobial peptides over longer time periods. However, while theabove studies utilized tissues that were incubated for at least 48 hr,improved antimicrobial activity was observed only in samples extractedfrom irradiated tissues for 2 or 4 hr.

In total, biochemical analysis of irradiated skin equivalents revealedthat the overall level of soluble protein was decreased, butantimicrobial peptide integrity was largely preserved. Peptidesextracted from skin equivalent tissues irradiated at 1 kGy were shown toreduce bacterial growth by up to 80% relative to control bacterialcultures. These results demonstrate the feasibility of maintaining thebiological activity of these terminally processed engineered skintissues.

Example 5 Storage of Irradiated Skin Equivalent Tissues

An analysis of the short-term storage capabilities of irradiated 9F1 and2D2 tissues stored on nutrient gels was undertaken. In these studies,refrigerated irradiated tissues were analyzed at 7 days and 37 days posttreatment, and assessed or assayed for tissue architecture andantimicrobial peptide activities. Overall tissue organization ofirradiated tissues was preserved during 37 days of refrigerated storage.At the dose levels examined, refrigerated tissues maintained both dermaland epidermal compartments. Within epidermal compartments, basal andspinous keratinocyte layers remain distinct, and cornified layers remainlargely unchanged. Minimal cellular damage within the tissue manifestsas intercellular spaces between keratinocytes in the basal andsuprabasal layers, and storage of tissues results in partial separationbetween the dermal and epidermal compartments. However, this was notedin tissues at all time periods examined.

Antimicrobial peptide activity: Samples were obtained from irradiated ornon-irradiated 9F1 and 2D2 tissues that had been stored at refrigeratedtemperatures for either 7 days or 37 days after irradiation.Antimicrobial activity assays were performed as above. Bacterial density(CFU/mL) was determined and data were normalized to bacterial culturesgrown in the presence of fresh serum-free growth medium. Results ofthese studies are shown in FIGS. 6 and 7 for 9F1 and 2D2 tissues,respectively. 9F1 tissues, irradiated with 1 kGy radiation and storedrefrigerated for 7 days, demonstrated an 86% reduction in bacterialgrowth relative to control bacterial cultures. In comparison,non-irradiated 9F1 tissues resulted in a 52% reduction in bacterialgrowth. Similarly, 1 kGy irradiated 2D2 tissues refrigerated for 7 daysresulted in a 72% reduction in bacterial growth, compared to a 57%reduction caused from non-irradiated 2D2 tissues. The antimicrobialactivities of 1 kGy-irradiated 9F1 and 2D2 tissues returned toapproximately those of non-irradiated tissues by 37 days of refrigeratedstorage, but still exhibited antimicrobial activity relative to controlcultures. Although all tissues treated with 5 kGy retained measurableantimicrobial activity at 7 days of post-irradiation storage, thisactivity was reduced upon prolonged storage. These data suggest thatirradiated tissues can provide antimicrobial activity that is detectableafter more than one month of refrigerated storage.

Example 6 Effects of Packaging Configuration

Tissues which had been processed in Example 2, above, were observed todetach from the underlying support membrane, resulting in folding andwrinkling of tissues. To circumvent this phenomenon, a packagingconfiguration was developed that prevented tissue movement. In thismodified configuration, tissues were removed from their inserts,transferred onto sterile, nonadherent gauze, and sealed in sterileplastic bags. After removal from the plastic bag, tissues showed noevidence of wrinkling or folding.

The effects of gamma irradiation on proteins have been describedelsewhere, and these effects are mediated in large part by free radicalsand reactive oxygen species generated by hydrolysis of water moleculeswithin the sample. Due to the high moisture content in nutrient gelchambers used for transport of the engineered tissue products, it wasanticipated that irradiated tissues packaged on nutrient gel chambers ordry nonadherent gauze during irradiation treatment would exhibitdifferent biochemical properties. Punch biopsies were obtained fromnon-irradiated and irradiated 2D2 and 9F1 tissues and submerged in 0.2mL sterile water. Biopsies were incubated at 37° C. for 24 hr,supernatants were collected, and eluted proteins were quantified by BCAassay. As shown in FIG. 8, total water-soluble protein decreased withincreasing radiation dose in tissues packaged on nutrient gel chambers,consistent with studies described above. In contrast, packaging ofirradiated tissues on nonadherent gauze restored protein accessibilityto approximately that of control levels. Packaging of tissues on gauzeprior to irradiation may therefore reduce the level of protein damage inirradiated tissue by presenting an environment that is less permissivefor protein crosslinking

Example 7 Freeze Drying of Engineered Skin Equivalents

Engineered skin equivalents are manufactured as described in Example 1.Upon completion, TRANSWELL inserts containing skin equivalents areaseptically transferred into plastic TRANSWELL dishes, covered, andplaced on the shelf of a VIRTIS Genesis Freeze Dryer (Gardiner, N.Y.)maintained at 20° C. Tissues are frozen by reducing the temperature from20° C. to −20° C. at a rate of −1.33° C. per minute at 2100 mT pressure;and from −20° C. to −60° C. at a rate of −0.67° C. per minute at 2100 mTpressure. Vacuum is applied to reduce pressure to 0 mT, and tissues arewarmed from −60° C. to 20° C. at +0.25° C. per minute. Drying iscompleted by holding samples at 0 mT at 20° C. for at least 16 hours.

Example 8 Vacuum Drying of Engineered Skin Equivalents

Engineered skin equivalents are manufactured as described in Example 1.TRANSWELL inserts containing skin equivalents are asepticallytransferred into plastic TRANSWELL dishes, covered, and placed on theshelf of a VIRTIS Genesis Freeze Dryer chamber maintained at 25° C.Chamber pressure is reduced at eight minute intervals to the following:900 mT, 830 mT, 760 mT, 690 mT, 620 mT, 550 mT, 490 mT, 430 mT, 370 mT,310 mT, 250 mT, 200 mT, 150 mT, 100 mT, 50 mT, 25 mT. Samples are heldat 25 mT for at least 16 hours to complete drying.

Example 9 Dry Mass of Engineered Skin Equivalents

Engineered skin equivalents are manufactured and wet tissue masses areobtained before and after freeze drying. Tissue mass obtained afterfreeze drying range from 11.7% to 13.7% of original wet tissue mass.(Table 1).

TABLE 1 Dry mass measurement of engineered skin equivalents Wet Mass DryMass % of Wet Tissue I.D. (mg) (mg) Mass 050508-1 1330.2 156.0 11.7050508-2 1035.5 122.4 11.8 050508-3 1204.4 165.2 13.7

Example 10 Irradiation of Dried Engineered Skin Equivalents

Skin equivalent tissues engineered for overexpression of the hostdefense peptide hCAP18/LL-37 are manufactured as described in Example 1,and dried as described in Examples 7 and 8. After drying, tissues areremoved from TRANSWELL inserts and heat-sealed in sterile plastic bags.Dried tissues are irradiated at Sterigenics as described in Example 2,at dose levels of 1, 5, or 25 kGy, followed by storage of tissues atroom temperature for up to two months. Overall tissue architecture isassessed by staining of histological sections with hematoxylin andeosin. Tissue viability is assessed by MTT assay as described in Example2. Tissue barrier function is assessed in dried irradiated tissues usingimpedance meter measurements. Specimens obtained from dried, irradiatedtissues are strained to failure under uniaxial tension and mechanicalproperties were determined. Antimicrobial peptide levels were quantifiedby ELISA of soluble extracts obtained from dried, irradiated tissues.

Histological analysis: Biopsies are obtained from fresh tissues, or fromtissues that had been freeze-dried or vacuum-dried at ambienttemperature and subsequently irradiated at one of three dose levels andstored at ambient temperature for up to two months. Specimens areprocessed for histological staining using hematoxylin/eosin in order tovisualize tissue architecture, and digital micrographs are obtained at400× magnification. Representative images of irradiated freeze-dried andvacuum-dried tissues are shown in FIG. 9 and FIG. 10, respectively.Freshly prepared specimens exhibit expected tissue architecture forengineered skin substitute tissues (FIGS. 9 and 10, panel 1).

Freeze-dried skin equivalent tissues subjected to radiation maintainnormal gross tissue architecture, with recognizable dermal and epidermalcomponents (FIG. 9). The dermal compartment exhibits structural changes,including compaction and partial delamination from the epidermalcompartment. Within the epidermal compartment, organization of thebasal, spinous, and stratum corneum layers are largely preserved.

Vacuum-dried irradiated engineered skin equivalents retain normal grosshistology (FIG. 10); including recognizable dermis, basal and spinouskeratinocyte layers, and stratum corneum. However, compaction of boththe dermal compartment is evident, resulting in a tissue which isthinner than unpreserved tissues. Increases in radiation dose did notintroduce further changes in the overall histology of vacuum-driedtissues.

Viability testing by MTT viability assay: Punch biopsies (8 mm diameter)were harvested from fresh tissues or from tissues that had beenfreeze-dried or vacuum-dried at ambient temperature and irradiated atone of three doses. Biopsies were processed, and metabolic activity wasquantified by measuring absorbance of the samples at 550 nm using aTECAN GENios plate reader (TECAN US, Durham, N.C.). Metabolic activitywas normalized to freshly prepared control tissues. As shown in FIG. 11,metabolic activity was reduced by up to 90% using a combination ofdrying and irradiation, consistent with the previously observedreduction in metabolic activity observed in irradiated tissues (Example2).

Tissue barrier function analysis: Barrier function was performed on ASTMdog-bone shaped specimens cut from engineered skin tissues that wereeither vacuum- or freeze-dried and exposed to one of three radiationdoses (1, 5, or 25 kGy). Specimens were punched from tissues followingdrying and packaged individually before storage or irradiation.

Dog-bone shaped specimens were removed from their packaging, rehydrated,and tested for barrier function. Briefly, specimens were placedepidermal-side-up onto TRANSWELL inserts, placed into dishes with 10 mlof media, and allowed to rehydrate (from the bottom up) for 1 hr at roomtemperature. The TRANSWELL inserts were transferred to wetted filterpapers and allowed to equilibrate for 45 min. Epidermal barrier functionin the grip regions of each specimen was quantified by measuring thesurface electrical capacitance of the tissue surface with a NOVADermaphase meter (NOVA Technology Corp, Portsmouth, N.H.), which is usedclinically to assess epidermal barrier function. Changes in theimpedance measurements over a 10 second measurement period reflectchanges in the hydration state of the tissue surface. Because increasedhydration results from passage of water through the stratum corneum, themagnitude of the change reflects the integrity of the epidermalpermeability barrier. Based on barrier function data collected from morethan 80 lots of StrataGraft® tissue, initial readings of <294 DPM andchanges of less than 658 DPM units are considered acceptable barrierfunction.

As shown in FIG. 12, the change in tissue surface electrical impedanceand initial DPM readings were similar for dried irradiated tissues andfreshly prepared engineered skin tissues. Both freshly prepared anddried irradiated tissues achieved epidermal barrier function that wasdeemed acceptable according to the historical data compiled forStrataGraft® skin tissue. Based on these results, it is anticipated thatirradiation of dried engineered skin tissue will not cause adverseeffects on the barrier function of the resultant product.

Tensile Strength Analysis—Following epidermal barrier testing, eachspecimen was submerged in 10 ml of PBS and allowed to rehydrate for atleast 1 additional hour. Following rehydration, specimen thickness wasmeasured in the gage region using a Mitutoyo thickness gauge. Tensilespecimens were then pulled to failure in uniaxial tension at a rate of100%/min (25 mm/min), with specimen hydration maintained by PBSrecirculation. Load and displacement data from each experiment wereexported to Microsoft Excel for analysis and data compilation. Data fromthese analyses are presented in FIG. 13.

Drying and irradiation of engineered skin equivalents resulted invariable effects on the mechanical properties of the tissues. Analysisof the mechanical testing results was complicated by the highvariability between tissue specimens, however several strong trends wereobserved. Dried tissues, regardless of drying method or irradiationdose, are unable to fully regain their pre-drying thickness followingrehydration, resulting in significantly thinner tissue specimens whencompared to fresh controls. From histology, this reduction appears tooccur mainly in the dermal layer, with epidermal thickness remainingrelatively constant. There is also a strong trend for drying to resultin a stiffer and more brittle tissue, with irradiation adding to thiseffect. This can be seen by both the increased initial modulus, as wellas the reduction in elongation at failure. Although some of the increasein modulus values can be attributed to the reduction in thickness(increasing the measured stress for a given increase in load), thicknessdifferences do not fully account for the differences between groups.

The degree of variability in the results makes it difficult to discernadditional effects with statistical certainty. Average tensile strengthdid not appear to be adversely affected by either drying or irradiationup to 25 kGy; however, irradiation may have caused a slight, butstatistically insignificant, decrease in the peak load.

Antimicrobial Peptide analysis: hCAP18-derived peptides from engineeredskin equivalents were quantified using a commercially available ELISAdetection kit. Soluble extracts were prepared by topically applyingserum-free culture medium to the surface of fresh or preserved tissues(0.16 ml serum-free medium per cm²), and equilibrating the tissues for 2hr. at 37° C. Extracts were used in an ELISA that detects intact hCAP18protein and posttranslationally processed LL37 metabolites. Samples werequantified relative to a standard curve of recombinant LL37 peptide andhCAP18 protein levels were expressed as ng protein per ml of tissueextract.

As shown in Table 2, freeze-dried and vacuum-dried tissues exhibitedlevels of extractable hCAP18-derived peptide greater than 75% of thoseobtained from freshly prepared engineered skin tissues.

TABLE 2 Quantification of hCAP18-derived Peptides in Dried Irradiatedengineered skin equivalents. Values are reported as mean concentration ±standard deviation of immunoreactive protein in tissue extracts from twoindependent tissues. Treatment Group hCAP18 protein (ng/ml) EG111008Fresh 97.4 ± 14  EG111008 Freeze-dried 0 kGy 73.1 ± 4.8 EG111008Freeze-dried 1 kGy  183 ± 4.8 EG111008 Freeze-dried 5 kGy  179 ± 2.0EG111008 Freeze-dried 25 kGy  120 ± 3.2 EG111008 Vacuum-dried 0 kGy 122± 10 EG111008 Vacuum-dried 1 kGy 120 ± 15 EG111008 Vacuum-dried 5 kGy77.7 ± 3.4 EG111008 Vacuum-dried 25 kGy 66.8 ± 7.0

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in tissueculture, molecular biology, biochemistry, or related fields are intendedto be within the scope of the following claims.

We claim:
 1. A method for treating a wound in a subject comprising: applying an isolated, non-viable, in vitro human skin equivalent to said wound in said subject under conditions such that said skin equivalent contacts said wound and said wound is treated, wherein the skin equivalent comprises organotypically cultured keratinocytes that have stratified into squamous epithelia; and cell viability in the skin equivalent has been eliminated.
 2. The method of claim 1, wherein said skin equivalent is applied to said wound temporarily.
 3. The method of claim 1, wherein said skin equivalent is sterile.
 4. The method of claim 3, wherein said sterile skin equivalent is irradiated.
 5. The method of claim 1, wherein said skin equivalent is dried.
 6. The method of claim 5, wherein said skin equivalent has a mass of less than 50% of the mass of a wet skin equivalent.
 7. The method of claim 6, wherein said skin equivalent, after rehydration, has an initial DPM value of from 20 to 300, and a DPM change value of from 5 to
 400. 8. The method of claim 6, wherein said skin equivalent, after rehydration, has a tensile strength of from 0.1 to 5.0 MPa.
 9. The method of claim 1, wherein said skin equivalent comprises Near-Diploid Immortalized Keratinocytes (NIKS) cells.
 10. The method of claim 9, wherein said NIKS cells comprise an exogenous nucleic acid sequence encoding an exogenous polypeptide.
 11. The method of claim 10, wherein said exogenous polypeptide is an antimicrobial polypeptide.
 12. The method of claim 11, wherein said antimicrobial polypeptide is selected from the group consisting of human beta-defensin 1, human beta-defensin 2, human beta-defensin 3, cathelicidin, and combinations thereof.
 13. The method of claim 12, wherein said antimicrobial polypeptide is provided in a quantity of from 10 to 500 ng of antimicrobial polypeptide per milliliter of a surface extraction solution. 